LRP5 Regulates Development of Lung Microvessels and Alveoli through the Angiopoietin-Tie2 Pathway

Angiogenesis is crucial for lung development. Although there has been considerable exploration, the mechanism by which lung vascular and alveolar formation is controlled is still not completely understood. Here we show that low-density lipoprotein receptor-related protein 5 (LRP5), a component of the Wnt ligand-receptor complex, regulates angiogenesis and alveolar formation in the lung by modulating expression of the angiopoietin (Ang) receptor, Tie2, in vascular endothelial cells (ECs). Vascular development in whole mouse lungs and in cultured ECs is controlled by LRP5 signaling, which is, in turn, governed by a balance between the activities of the antagonistic Tie2 ligands, Ang1 and Ang2. Under physiological conditions when Ang1 is dominant, LRP5 knockdown decreases Tie2 expression and thereby, inhibits vascular and alveolar development in the lung. Conversely, when Ang2 dominates under hyperoxia treatment in neonatal mice, high LRP5 and Tie2 expression suppress angiogenesis and lung development. These findings suggest that the LRP5-Tie2-Ang signaling axis plays a central role in control of both angiogenesis and alveolarization during postnatal lung development, and that deregulation of this signaling mechanism might lead to developmental abnormalities of the lung, such as are observed in bronchopulmonary dysplasia (BPD).     

Expression of the receptor tyrosine kinase Tie2 in neoplastic glial cells is associated with integrin beta1-dependent adhesion to the extracellular matrix

The abnormal function of tyrosine kinase receptors is a hallmark of malignant gliomas. Tie2 receptor tyrosine kinase is a specific endothelial cell receptor whose function is positively regulated by angiopoietin 1 (Ang1). Recently, Tie2 has also been found in the nonvascular compartment of several tumors, including leukemia as well as breast, gastric, and thyroid cancers. There is, however, little information on the function of the Ang1/Tie2 pathway in the non-stromal cells within human tumors. We found that surgical glioblastoma specimens contained a subpopulation of Tie2+/CD31- and Tie2+/GFAP+ cells, suggesting that Tie2 is indeed expressed outside the vascular compartment of gliomas. Furthermore, analysis of a tissue array consisting of 116 human glioma samples showed that Tie2 expression in the neoplastic glial cells was significantly associated with progression from a lower to higher grade. Importantly, Ang1 stimulation of Tie2+ glioma cells resulted in increased adherence of the cells to collagen I and IV, suggesting that Tie2 regulates glioma cell adhesion to the extracellular matrix. Conversely, the down-regulation of Tie2 levels by small interference RNA or the addition of soluble Tie2 abrogated the Ang1-mediated effect on cell adhesion. In studying the expression of cell adhesion molecules, we found that Tie2 activation was related to the up-regulation of integrin beta1 levels and the formation of focal adhesions. These results, together with the reported fact that malignant gliomas express high levels of Ang1, suggest the existence of an autocrine loop in malignant gliomas and that a Tie2-dependent pathway modulates cell-to-extracellular matrix adhesion, providing new insights into the highly infiltrative phenotype of human gliomas.     

Mechanisms of angiogenesis

Tissue activity of angiogenesis depends on the balance of many stimulating or inhibiting factors. The key signaling system that regulates proliferation and migration of endothelial cells forming the basis of any vessel are vascular endothelium growth factors (VEGF) and their receptors. The VEGF-dependent signaling system is necessary for formation of the embryonic vascular system. Neoangiogenesis during tumor growth is also associated with activation of this signaling system. The biological significance of the effect of such system on the cells depends on the content in tissue of various factors of the VEGF family and their receptors, while in the case of VEGFA it is defined by the ratio of different isoforms of this growth factor. A number of other signaling systems are also involved in regulation of the main steps of vessel formation. The signaling system Dll4/Notch regulates selection of endothelial cells for beginning of angiogenic expansion by endowing particular properties to endothelial cells leading in this process. An important step in vessel stabilization and maturation is vascular wall formation. Signaling system PDGFB/PDGFRbeta as well as angiopoietins Ang1, Ang2, and their receptor Tie2 are involved in recruiting mural cells (pericytes and smooth muscle cells). Identification of key molecules involved in the regulation of angiogenesis may provide new possibilities for development of drugs suitable for inhibition of angiogenesis or its stimulation in various pathologies.     

Differential Expression of Tie2 Receptor and VEGFR2 by Endothelial Clones Derived from Isolated Bovine Mononuclear Cells

The purpose of these experiments was to evaluate the expression of endothelial markers, such as Tie2 and VEGFR2 in endothelial cells derived from blood mononuclear endothelial progenitor cells. Bovine mononuclear cells were isolated using separation by centrifugation and were grown in endothelial specific media supplemented with growth factors. Isolation of the whole cell population of mononuclear cells (MNC) from bovine peripheral blood gave rise to progenitor-like cells (CD45⁻) that, although morphologically similar, have different phenotypes revealed by expression of endothelial specific markers Tie2 and VEGFR2. Plating of MNCs on collagen and fibronectin gave rise to more colonies than non-coated dishes. Occasional colonies from MNC isolations had a mural cell phenotype, negative for Tie2 and VEGFR2 but positive for smooth muscle actin and PDGFRβ. Although cells expressing high levels of VEGFR2 and low levels of Tie2, and vice versa were both able to form cords on Matrigel, cells with higher expression of Tie2 migrate faster in a scratch assay than ones with lower expression of Tie2. When these different clones of cells were introduced in mice through tail vein injections, they retained an ability to home to angiogenesis occurring in a subcutaneous Matrigel plug, regardless of their Tie2/VEGFR2 receptor expression patterns, but cells with high VEGFR2/low Tie2 were more likely to be CD31 positive. Therefore, we suggest that active sites of angiogenesis (such as wounds, tumors, etc.) can attract a variety of endothelial cell precursors that may differentially express Tie2 and VEGFR2 receptors, and thus affect our interpretation of EPCs as biomarkers or therapies for vascular disease.     

TIE2-high cervical cancer cells promote tumor angiogenesis by upregulating TIE2 and VEGFR2 in endothelial cells

Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (TIE2), the receptor for angiopoietins, has been found highly expressed in cervical cancer and associated with poor prognosis. However, the potential role of tumoral TIE2 in cervical cancer angiogenesis and the underlying mechanisms remain unexplored. Here, based on multicolor immunofluorescence of 64 cervical cancer tissues, we found that TIE2 level in cervical cancer cells was positively related to shorter survival and higher microvessel density in tumor. In vitro and in vivo experiments verified that TIE2-high cervical cancer cells could promote tumor angiogenesis. TIE2-high tumor cells induced an amplified expression of TIE2 and vascular endothelial growth factor receptor 2(VEGFR2) in HUVECs to promote angiogenesis via TIE2 -AKT/MAPK signals, which could be reversed or partially reversed by TIE2, AKT or MAPK inhibitors and activated by angiopoietin-1 and angiopoietin-2. In conclusion, TIE2-high cervical cancer cells promote tumor angiogenesis by upregulating TIE2 and VEGFR2 in endothelial cells via TIE2-AKT/MAPK axis inside tumor cells.     

Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis

The VEGF/VPF (vascular endothelial growth factor/vascular permeability factor) ligands and receptors are crucial regulators of vasculogenesis, angiogenesis, lymphangiogenesis and vascular permeability in vertebrates. VEGF-A, the prototype VEGF ligand, binds and activates two tyrosine kinase receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). VEGFR1, which occurs in transmembrane and soluble forms, negatively regulates vasculogenesis and angiogenesis during early embryogenesis, but it also acts as a positive regulator of angiogenesis and inflammatory responses, playing a role in several human diseases such as rheumatoid arthritis and cancer. The soluble VEGFR1 is overexpressed in placenta in preeclampsia patients. VEGFR2 has critical functions in physiological and pathological angiogenesis through distinct signal transduction pathways regulating proliferation and migration of endothelial cells. VEGFR3, a receptor for the lymphatic growth factors VEGF-C and VEGF-D, but not for VEGF-A, regulates vascular and lymphatic endothelial cell function during embryogenesis. Loss-of-function variants of VEGFR3 have been identified in lymphedema. Formation of tumor lymphatics may be stimulated by tumor-produced VEGF-C, allowing increased spread of tumor metastases through the lymphatics. Mapping the signaling system of these important receptors may provide the knowledge necessary to suppress specific signaling pathways in major human diseases.     

Molecular control of angiopoietin signalling

The angiopoietins act through the endothelial receptor tyrosine kinase Tie2 to regulate vessel maturation in angiogenesis and control quiescence and stability of established vessels. The activating ligand, Ang1 (angiopoietin-1), is constitutively expressed by perivascular cells, and the ability of endothelial cells to respond to the ligand is controlled at the level of the Ang1 receptor. This receptor interacts with the related protein Tie1 on the cell surface, and Tie1 inhibits Ang1 signalling through Tie2. The responsiveness of endothelium to Ang1 is determined by the relative levels of Tie2 and the inhibitory co-receptor Tie1 in the cells. Tie1 undergoes regulated ectodomain cleavage which is stimulated by a range of factors including VEGF (vascular endothelial growth factor), inflammatory cytokines and changes in shear stress. Ectodomain cleavage of Tie1 relieves inhibition of Tie2 and enhances Ang1 signalling. This mechanism regulates Ang1 signalling without requiring changes in the level of the ligand and allows Ang1 signalling to be co-ordinated with other signals in the cellular environment. Regulation of signalling at the level of receptor responsiveness may be an important adaptation in systems in which an activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal.     

Expression of genes involved in vascular development and angiogenesis in endothelial cells of adult lung

Profiling gene expression in endothelial cells advances the understanding of normal vascular physiology and disease processes involving angiogenesis. However, endothelial cell purification has been challenging because of the difficulty of isolating cells and their low abundance. Here we examine gene expression in endothelial cells freshly isolated from lung capillaries after in vivo labeling with fluorescent cationic liposomes and purification by fluorescence-activated cell sorting (FACS). Of the 39,000 genes and expressed sequence tags evaluated on custom oligonucleotide arrays, 555 were enriched in endothelial cell fraction. These included familiar endothelial cell-associated genes such as VEGF, VEGF receptor (VEGFR)-1, VEGFR-2, angiopoietin-2, Tie1, Tie2, Edg1 receptor, VE-cadherin, claudin 5, connexin37, CD31, and CD34. Also enriched were genes in semaphorin/neuropilin (Sema3c and Nrp1), ephrin/Eph (ephrin A1, B1, B2, and EphB4), delta/notch (Hey1, Jagged 2, Notch 1, Notch 4, Numb, and Siah1b), and Wingless (Frizzled-4 and Tle1) signaling pathways involved in vascular development and angiogenesis. Expression of representative genes in alveolar capillary endothelial cells was verified by immunohistochemistry. Such expression reflects features that endothelial cells of normal lung capillaries have in common with embryonic and growing blood vessels. About half of the enriched genes, including exostosin 2, lipocalin 7, phospholipid scramblase 2, pleckstrin 2, protocadherin 1, Ryk, scube 1, serpinh1, SNF-related kinase, and several tetraspanins, had little or no previous association with endothelial cells. This approach can readily be used to profile genes expressed in blood vessels in tumors, chronic inflammation, and other sites in which endothelial cells avidly take up cationic liposomes.     

Vascular endothelial growth factor activates the Tie family of receptor tyrosine kinases

The ability of cells to respond appropriately to changes in their environment requires integration and cross-talk between relevant signalling pathways. The vascular endothelial growth factor (VEGF) and angiopoietin families of ligands are key regulators of blood vessel formation. VEGF binds to receptor tyrosine kinases of the VEGF-receptor family to activate signalling pathways leading to endothelial migration, proliferation and survival whereas the angiopoietins interact with the Tie receptor tyrosine kinases to control vessel stability, survival and maturation. Here we show that VEGF can also activate the angiopoietin receptor Tie2. Activation of human endothelial cells with VEGF caused a four-fold stimulation of tyrosine phosphorylation of Tie2. This stimulation was not due to VEGF-induction of Tie2 ligands as soluble ligand binding domain of Tie2 failed to inhibit VEGF activation of the receptor. Immunoprecipitation analysis demonstrated no physical interaction between VEGF receptors and Tie2. However Tie2 does interact with the related receptor tyrosine kinase Tie1 and this receptor was found to be essential for VEGF activation of Tie2. VEGF stimulated proteolytic cleavage of Tie1 generating a truncated Tie1 intracellular domain. Similarly, phorbol ester also both stimulated Tie1 truncation and activated Tie2 phosphorylation. Inhibition of Tie1 cleavage with the metalloprotease inhibitor TAPI-2 suppressed VEGF- and phorbol ester-induced phosphorylation of Tie2. Truncated Tie1 formed in response to VEGF was also found to be tyrosine phosphorylated and this was independent of Tie2, though Tie2 could enhance Tie1 intracellular domain phosphorylation. Together these data demonstrate that VEGF activates Tie2 via a mechanism involving proteolytic cleavage of the associated tyrosine kinase Tie1 leading to trans-phosphorylation of Tie2. This novel mechanism of receptor tyrosine kinase activation is likely to be important in integrating signalling between two of the key receptor groups regulating angiogenesis.     

Differential function of Tie2 at cell-cell contacts and cell-substratum contacts regulated by angiopoietin-1

Tie2 belongs to the receptor tyrosine kinase family and functions as a receptor for Angiopoietin-1 (Ang1). Gene-targeting analyses of either Ang1 or Tie2 in mice reveal a critical role of Ang1-Tie2 signalling in developmental vascular formation. It remains elusive how the Tie2 signalling pathway plays distinct roles in both vascular quiescence and angiogenesis. We demonstrate here that Ang1 bridges Tie2 at cell-cell contacts, resulting in trans-association of Tie2 in the presence of cell-cell contacts. In clear contrast, in isolated cells, extracellular matrix-bound Ang1 locates Tie2 at cell-substratum contacts. Furthermore, Tie2 activated at cell-cell or cell-substratum contacts leads to preferential activation of Akt and Erk, respectively. Microarray analyses and real-time PCR validation clearly show the differential gene expression profile in vascular endothelial cells upon Ang1 stimulation in the presence or absence of cell-cell contacts, implying downstream signalling is dependent upon the spatial localization of Tie2.     

BMPs promote proliferation and migration of endothelial cells via stimulation of VEGF-A/VEGFR2 and angiopoietin-1/Tie2 signalling

The differentiation, growth, and survival of endothelial cells (ECs) are regulated by multiple signalling pathways, such as vascular endothelial growth factors (VEGFs) and angiopoietins through their receptor tyrosine kinases, VEGF receptor (VEGFR) 2 and Tie2, respectively. Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)-beta family, have been implicated in the development and maintenance of vascular systems. However, their effects on EC proliferation remain to be elucidated. In the present study, we show that BMPs induce the proliferation and migration of mouse embryonic stem cell (ESC)-derived endothelial cells (MESECs) and human microvascular endothelial cells (HMECs). Addition of BMP-4 to culture induced significant proliferation and migration of both types of ECs. BMP-4 also increased the expression and phosphorylation of VEGFR2 and Tie2. These findings suggest that BMP signalling activates endothelium via activation of VEGF/VEGFR2 and Angiopoietin/Tie2 signalling.     

MEKK3 is required for endothelium function but is not essential for tumor growth and angiogenesis

Mitogen-activated protein kinase kinase kinase 3 (MEKK3) plays an essential role in embryonic angiogenesis, but its role in tumor growth and angiogenesis is unknown. In this study, we further investigated the role of MEKK3 in embryonic angiogenesis, tumor angiogenesis, and angiogenic factor production. We found that endothelial cells from Mekk3-deficient embryos showed defects in cell proliferation, apoptosis, and interactions with myocardium in the heart. We also found that MEKK3 is required for angiopoietin-1 (Ang1)-induced p38 and ERK5 activation. To study the role of MEKK3 in tumor growth and angiogenesis, we established both wild-type and Mekk3-deficient tumor-like embryonic stem cell lines and transplanted them subcutaneously into nude mice to assess their ability to grow and induce tumor angiogenesis. Mekk3-deficient tumors developed and grew similarly as control Mekk3 wild-type tumors and were also capable of inducing tumor angiogenesis. In addition, we found no differences in the production of VEGF in Mekk3-deficient tumors or embryos. Taken together, our results suggest that MEKK3 plays a critical role in Ang1/Tie2 signaling to control endothelial cell proliferation and survival and is required for endothelial cells to interact with the myocardium during early embryonic development. However, MEKK3 is not essential for tumor growth and angiogenesis. cardiovascular; mitogen-activated protein kinase; embryonic development     

The angiopoietins and Tie2/Tek: adding to the complexity of cardiovascular development

The expansion or remodelling of pre-existing blood vessels, known as angiogenesis, by either nascent sprouting, intercalated or intussusceptive growth is a highly regulated process. Angiogenesis is critical not only during normal embryonic vascular development, but also in the progression of several diseases, including cancer, psoriasis, and diabetes. Mouse molecular genetic experiments have shown that the angiopoietins and their receptor Tie2/Tek are indispensable for embryonic vessel development. The importance of the angiopoietin-signalling pathway has also been shown to extend beyond development, into in vitro and in vivo experimental models of angiogenic growth. Currently the precise role of the angiopoietins remains unclear. However, what is emerging from genetic, xenograft transplant, histochemical and cell culture experiments are that the response of endothelial cells to angiopoietins appears to be context and endothelial cell type specific.     

Tie1 regulates the Tie2 agonistic role of angiopoietin-2 in human lymphatic endothelial cells

Although Angiopoietin (Ang) 2 has been shown to function as a Tie2 antagonist in vascular endothelial cells, several recent studies on Ang2-deficient mice have reported that, like Ang1, Ang2 acts as a Tie2 agonist during in vivo lymphangiogenesis. However, the mechanism governing the Tie2 agonistic activity of Ang2 in lymphatic endothelial cells has not been investigated. We found that both Ang1 and Ang2 enhanced the in vitro angiogenic and anti-apoptotic activities of human lymphatic endothelial cells (HLECs) through the Tie2/Akt signaling pathway, while only Ang1 elicited such effects in human umbilical vein vascular endothelial cells (HUVECs). This Tie2-agonistic effect of Ang2 in HLECs resulted from low levels of physical association between Tie2 and Tie1 receptors due to a reduced level of Tie1 expression in HLECs compared to HUVECs. Overexpression of Tie1 and the resulting increase in formation of Tie1/Tie2 heterocomplexes in HLECs completely abolished Ang2-mediated Tie2 activation and the subsequent cellular responses, but did not alter the Ang1 function. This inhibitory role of Tie1 in Ang2-induced Tie2 activation was also confirmed in non-endothelial cells with adenovirus-mediated ectopic expression of Tie1 and/or Tie2. To our knowledge, this study is the first to describe how Ang2 acts as a Tie2 agonist in HLECs. Our results suggest that the expression level of Tie1 and its physical interaction with Tie2 defines whether Ang2 functions as a Tie2 agonist or antagonist, thereby determining the context-dependent differential endothelial sensitivity to Ang2.     

Intra and extravascular transmembrane signalling of angiopoietin-1-Tie2 receptor in health and disease

Angiopoietin-1 (Ang-1) is the primary agonist for Tie2 tyrosine kinase receptor (Tie2), and the effect of Ang-1-Tie2 signalling is context-dependent. Deficiency in either Ang-1 or Tie2 protein leads to severe microvascular defects and subsequent embryonic lethality in murine model. Tie2 receptors are expressed in several cell types, including endothelial cells, smooth muscle cells, fibroblasts, epithelial cells, monocytes, neutrophils, eosinophils and glial cells. Ang-1-Tie2 signalling induces a chemotactic effect in smooth muscle cells, neutrophils and eosinophils, and induces differentiation of mesenchymal cells to smooth muscle cells. Additionally, this signalling pathway induces the secretion of serotonin, matrix metalloproteinases (MMPs) and plasmin. Ang-1 inhibits the secretion of tissue inhibitor of matrix metalloproteinase (TIMPs). Aberrant expression and activity of Tie2 in vascular and non-vascular cells may result in the development of rheumatoid arthritis, cancer, hypertension and psoriasis. Ang-1 has an anti-inflammatory effect, when co-localized with vascular endothelial growth factor (VEGF) in the vasculature. Thus, Ang-1 could be potentially important in the therapy of various pathological conditions such as pulmonary hypertension, arteriosclerosis and diabetic retinopathy. In this article, we have summarized and critically reviewed the pathophysiological role of Ang-1-Tie2 signalling pathway.     

Tyrosine 1101 of Tie2 Is the Major Site of Association of p85 and Is Required for Activation of Phosphatidylinositol 3-Kinase and Akt

Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2’s role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.     

Osteoblast-specific Angiopoietin 1 overexpression increases bone mass

Although osteoblasts express the angiogenic protein Angiopoietin 1 (Ang1), the role of Ang1 in bone formation remains largely unknown. Here we report that Ang1 overexpression in osteoblasts driven by the osteoblast-specific 2.3 kb alpha 1 type 1 collagen promoter results in increased bone mass in vivo. In Ang1-transgenic mice (Ang1-Tg), bone volume and bone parameters increased significantly compared with wild-type littermates, although the Ang1 receptor, Tie2 was not expressed in osteoblasts. Tie2 is primarily expressed in vascular endothelial cells, and Ang1-Tie2 signaling is reportedly crucial for angiogenesis. We found that the number of vascular endothelial cells was significantly elevated in Ang1-Tg mice compared with that of wild-type littermates, an increase accompanied by increased alkaline-phosphatase activity, a marker of osteoblast activation. The number of osteoclasts in the bone of Ang1-Tg mice did not differ from wild-type littermates. These results indicate that angiogenesis induced by Ang1 expressed in osteoblasts is coupled with osteogenesis.     

Gln-362 of Angiopoietin-2 Mediates Migration of Tumor and Endothelial Cells through Association with α5β1 Integrin

Angiopoietin-2 (Ang-2) not only regulates angiogenesis by binding to its well known receptor Tie2 on endothelial cells but also controls sprouting of Tie2-negative angiogenic endothelial cells and invasion of Tie2-negative non-endothelial cells by binding to integrins. However, the molecular mechanism of the Ang-2/integrin association has been unclear. In this study, we found that the Gln-362 residue of Ang-2 was essential for binding to α5β1 integrin. A Q362E Ang-2 mutant, which still bound to Tie2, failed to associate with α5β1 integrin and was unable to activate the integrin downstream signaling of focal adhesion kinase. In addition, unlike wild-type Ang-2, the Q362E Ang-2 mutant was defective in mediating invasion of Tie2-negative glioma or Tie2-positive endothelial cells. Furthermore, the tailpiece domain of the α5 subunit in α5β1 integrin was critical for binding to Ang-2. Taken together, these results provide a novel insight into the mechanism of integrin regulation by Ang-2, which contributes to tumor invasion and endothelial cell migration in a Tie2-independent manner.